A genetic and bioinformatic analysis of Streptomyces coelicolor genes containing TTA codons, possible targets for regulation by a developmentally significant tRNA

The rarest codon in the high G+C genome of Streptomyces coelicolor is TTA, corresponding in mRNA to the UUA codon that is recognized by a developmentally important tRNA encoded by the bldA gene. There are 145 TTA-containing genes in the chromosome of S. coelicolor. Only 42 of these are represented in the genome of Streptomyces avermitilis, among which only 12 have a TTA codon in both species. The TTA codon is less represented in housekeeping genes and orthologous genes, and is more represented in functional-unknown, extrachromosomal or weakly expressed genes. Twenty one TTA-containing chromosomal genes in S. coelicolor were disrupted, including 12 of the 42 genes that are common to both S. avermitillis and S. coelicolor. None of the mutant strains showed any obvious phenotypic differences from the wild-type strain under tested conditions. Possible reasons for this, and the role and evolution of the observed distribution of TTA codons among Streptomyces genes were discussed.     

Evolutionary flux of potentially <Emphasis Type="Italic">bldA</Emphasis>-dependent <Emphasis Type="Italic">Streptomyces</Emphasis> genes containing the rare leucine codon TTA

Previous studies have shown that one of the six leucine codons, UUA, is rare in Streptomyces, and that, while the gene for the UUA-specific tRNA, bldA, can generally be inactivated in diverse streptomycetes without impairing vegetative growth, bldA mutants are typically defective in reproductive aerial growth and in antibiotic production. Here, four complete genome sequences and 143 gene clusters for antibiotic biosynthesis from diverse streptomycetes were analysed in order to evaluate the evolution and function of genes whose possession of TTA codons makes them dependent on bldA. It was deduced that the last common ancestor of the four sequenced genomes, possibly 220 million years ago, already possessed the bldA system, together with perhaps 200 TTA-containing target genes. Some 33 of these genes are retained by the modern descendants, though only three of them retain a TTA in all occurrences. Nearly all of these 33, as well as many of the TTA-containing genes with orthologues in two or three of the four genomes, have the same location on the chromosomes as in their common ancestor. However, the majority of TTA-containing genes (61% overall in the four genomes) are species-specific, and were probably acquired by comparatively recent horizontal gene transfer. Most of these genes are of unknown function, and it is likely that many of them confer specialised ecological benefits. On the other hand, one class of species-specific, functionally recognisable, horizontally acquired genes--the gene clusters for antibiotic production--very often contain TTA codons; and nearly half of them have TTA codons in their pathway-specific regulatory genes.     

The Use of the Rare TTA Codon in Streptomyces Genes: Significance of the Codon Context?

Streptomycetes, Gram-positive bacteria with huge and GC-rich genomes provide an ample example of codon usage bias taken to the extreme. Particularly, in all sequenced to date streptomycete genomes leucyl codon TTA is the rarest one. It is present (usually once or twice) in 70–200 out of 7000–8000 coding sequences that make up a typical streptomycete genome. tRNA^Leu_UAA of streptomycetes, encoded by the bldA gene, has been shown to be present in mature form only after the onset of morphological differentiation and activation of secondary metabolism. Consequently, during the early stages of cell growth, the translation of genes carrying the TTA codon can be interrupted due to the absence of tRNA^Leu_UAA. Several reports show that mutations of TTA to synonymous codons in certain genes indeed relieve their expression from bldA dependence. However, the deletion of bldA does not always arrest the expression of TTA-containing genes. The nucleotides T/C downstream of TTA were suggested, in 2002, to favor TTA mistranslation. We tested this hypothesis using sizable datasets derived from individual Streptomyces genome and a subset of TTA+ genes for secondary metabolism known for their active expression. Our results revealed nucleotide biases downstream of NNA codons family, such as the preference for C and the avoidance of A. Yet, none of the observed biases was sufficient to claim a special case for TTA codon. Hence, the issue of codon context and TTA codon mistranslation in Streptomyces deserves further elaboration.Electronic supplementary materialThe online version of this article (10.1007/s12088-020-00902-6) contains supplementary material, which is available to authorized users.     

The use of a rare codon specifically during development?

A range of circumstantial evidence suggests that in Streptomyces spp., genes required for vegetative growth do not contain the leucine codon TTA. Instead, the codon seems to be confined to a few genes necessary during differentiation, when the colonies begin to produce aerial hyphae and antibiotics. Thus, mutations in bldA, the structural gene for tRNATTALeu, do not retard vegetative growth, but they prevent normal aerial mycelium and antibiotic production. Most of the known TTA-containing genes specify regulatory or resistance proteins associated with antibiotic-production clusters. Possibly the ability to translate the UUA codons in mRNA from such genes is confined to late stages of colony development. Factors that might have contributed to the evolution of this unusual situation are discussed.     

Codon usage bias in human cytomegalovirus and its biological implication

Human cytomegalovirus (HCMV) infection, a worldwide contagion, causes a serious disorder in infected individuals. Analysis of codon usage can reveal much molecular information about this virus. The effective number of codon (ENC) values, relative synonymous codon usage (RSCU) values, codon adaptation index (CAI), and nucleotide contents was investigated in approximately 160 coding sequences (CDS) among 17 human cytomegalovirus genomes using the software CodonW. Linear regression analysis and logistic regression were performed to explore the preliminary data. The results showed that, overall, HCMV genomes had low codon usage bias (mean ENC = 47.619). However, the ENC of individual CDS varied widely and was distributed unevenly between host-related genes and viral-self-function genes (P = 0.002, odds ratio (OR) = 3.194), as did the GC content (P = 0.016, OR = 2.178). The ENC values correlated with CAI, GC content, and the nucleotide composing at the 3rd codon position (GC3s) (P < 0.001). There was a significant variation in the codon preference that depended on the RSCU data. The predicted ENC curve suggested that mutational pressure, rather than natural selection, was one of the main factors that determined the codon usage bias in HCMV. Among 123 genes with known function, the genes related to viral self-replication and viral–host interaction showed different ENC and CAI values, and GC and GC3s contents. In conclusion, the detailed codon usage bias theoretically revealed information concerning HCMV evolution and could be a valuable additional parameter for HCMV gene function research.     

Comprehensive analysis of prokaryotic mechanosensation genes: their characteristics in codon usage.

In the present study, we examined GC nucleotide composition, relative synonymous codon usage (RSCU), effective number of codons (ENC), codon adaptation index (CAI) and gene length for 308 prokaryotic mechanosensitive ion channel (MSC) genes from six evolutionary groups: Euryarchaeota, Actinobacteria, Alphaproteobacteria, Betaproteobacteria, Firmicutes, and Gammaproteobacteria. Results showed that: (1) a wide variation of overrepresentation of nucleotides exists in the MSC genes; (2) codon usage bias varies considerably among the MSC genes; (3) both nucleotide constraint and gene length play an important role in shaping codon usage of the bacterial MSC genes; and (4) synonymous codon usage of prokaryotic MSC genes is phylogenetically conserved. Knowledge of codon usage in prokaryotic MSC genes may benefit from the study of the MSC genes in eukaryotes in which few MSC genes have been identified and functionally analysed.     

Comparative studies on codon usage pattern of chloroplasts and their host nuclear genes in four plant species

A detailed comparison was made of codon usage of chloroplast genes with their host (nuclear) genes in the four angiosperm speciesOryza sativa, Zea mays, Triticum aestivum andArabidopsis thaliana. The average GC content of the entire genes, and at the three codon positions individually, was higher in nuclear than in chloroplast genes, suggesting different genomic organization and mutation pressures in nuclear and chloroplast genes. The results of Nc-plots and neutrality plots suggested that nucleotide compositional constraint had a large contribution to codon usage bias of nuclear genes inO. sativa, Z. mays, andT. aestivum, whereas natural selection was likely to be playing a large role in codon usage bias in chloroplast genomes. Correspondence analysis and chi-test showed that regardless of the genomic environment (species) of the host, the codon usage pattern of chloroplast genes differed from nuclear genes of their host species by their AU-richness. All the chloroplast genomes have predominantly A- and/or U-ending codons, whereas nuclear genomes have G-, C- or U-ending codons as their optimal codons. These findings suggest that the chloroplast genome might display particular characteristics of codon usage that are different from its host nuclear genome. However, one feature common to both chloroplast and nuclear genomes in this study was that pyrimidines were found more frequently than purines at the synonymous codon position of optimal codons.     

A general model of codon bias due to GC mutational bias

Background

In spite of extensive research on the effect of mutation and selection on codon usage, a general model of codon usage bias due to mutational bias has been lacking. Because most amino acids allow synonymous GC content changing substitutions in the third codon position, the overall GC bias of a genome or genomic region is highly correlated with GC3, a measure of third position GC content. For individual amino acids as well, G/C ending codons usage generally increases with increasing GC bias and decreases with increasing AT bias. Arginine and leucine, amino acids that allow GC-changing synonymous substitutions in the first and third codon positions, have codons which may be expected to show different usage patterns.

Principal Findings

In analyzing codon usage bias in hundreds of prokaryotic and plant genomes and in human genes, we find that two G-ending codons, AGG (arginine) and TTG (leucine), unlike all other G/C-ending codons, show overall usage that decreases with increasing GC bias, contrary to the usual expectation that G/C-ending codon usage should increase with increasing genomic GC bias. Moreover, the usage of some codons appears nonlinear, even nonmonotone, as a function of GC bias. To explain these observations, we propose a continuous-time Markov chain model of GC-biased synonymous substitution. This model correctly predicts the qualitative usage patterns of all codons, including nonlinear codon usage in isoleucine, arginine and leucine. The model accounts for 72%, 64% and 52% of the observed variability of codon usage in prokaryotes, plants and human respectively. When codons are grouped based on common GC content, 87%, 80% and 68% of the variation in usage is explained for prokaryotes, plants and human respectively.

Conclusions

The model clarifies the sometimes-counterintuitive effects that GC mutational bias can have on codon usage, quantifies the influence of GC mutational bias and provides a natural null model relative to which other influences on codon bias may be measured.     

香樟转录组基因密码子偏好性分析

为了解香樟基因密码子偏好性,该文以NCBI网站中香樟转录组数据为材料,利用生物信息学手段评价转录组数据质量,选取高质量数据的转录组,去除低质量序列,组装转录组,预测基因结构,再利用自编perl脚本提取以AUG开头的基因序列37 Mb序列34 931个基因,进一步利用CodonW分析基因密码子偏好性。结果表明:GC含量的变化范围为0.273～0.742,均值为0.452; ENC的范围为26.29～61.00,均值为52.76; CAI的范围为0.064～0.401,均值为0.199; RSCU值大于1的密码子数目为27个,其中以U或A结尾的有22个; 中性分析表明,小部分基因在对角线上,大多数基因偏离对角线; ENC-plot分析表明小部分基因在标准曲线上,大多数基因偏离标准曲线。上述研究结果表明,香樟基因的密码子偏好性比较弱,密码子常以A/U结尾; 突变和选择两者都在密码子偏好中起作用,而选择作用更大; 最终确定了GUU、CAG、GAA、UCU、GCU、GGU为最优密码子,通过对目标基因密码子的校正,提高表达效率,从而为利用基因工程技术改良香樟重要性状奠定了基础。     

Variation in G + C-content and codon choice: differences among synonymous codon groups in vertebrate genes.

The relationship between G + C-content and codon usage in genes of human, mus, rat, bovine and chicken nuclear genomes was investigated. Correlation and lineal regression analyses were carried out on plots that related the frequency of each codon within each synonymous codon group to the G + C-content of the coding sequence as a whole. Under GC pressure, in most of the quartet codon groups there is a preferential choice of the C-ending codon, except in leucine and valine codon groups where the choice of the G-ending codon is preferred. Among ducts, the choice of codons specifying phenylalanine and glutamate shows the strongest dependence on G + C-content. The relationship found between G + C-content and codon usage in these genomes correlate with taxonomic distance.     

Analysis of synonymous codon usage patterns in different plant mitochondrial genomes

Codon usage in mitochondrial genome of the six different plants was analyzed to find general patterns of codon usage in plant mitochondrial genomes. The neutrality analysis indicated that the codon usage patterns of mitochondrial genes were more conserved in GC content and no correlation between GC12 and GC3. T and A ending codons were detected as the preferred codons in plant mitochondrial genomes. The Parity Rule 2 plot analysis showed that T was used more frequently than A. The EN_C-plot showed that although a majority of the points with low EN_C values were lying below the expected curve, a few genes lied on the expected curve. Correspondence analysis of relative synonymous codon usage yielded a first axis that explained only a partial amount of variation of codon usage. These findings suggest that natural selection is likely to be playing a large role in codon usage bias in plant mitochondrial genomes, but not only natural selection but also other several factors are likely to be involved in determining the selective constraints on codon bias in plant mitochondrial genomes. Meantime, 1 codon (P. patens), 6 codons (Z. mays), 9 codons (T. aestivum), 15 codons (A. thaliana), 15 codons (M. polymorpha) and 15 codons (N. tabacum) were defined as the preferred codons of the six plant mitochondrial genomes.     

Changes in the extracellular proteome caused by the absence of the bldA gene product, a developmentally significant tRNA, reveal a new target for the pleiotropic regulator AdpA in Streptomyces coelicolor

The extracellular proteome of Streptomyces coelicolor grown in a liquid medium was analyzed by using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time of flight peptide mass fingerprint analysis. Culture supernatants became protein rich only after rapid growth had been completed, supporting the idea that protein secretion is largely a stationary phase phenomenon. Out of about 600 protein spots observed, 72 were characterized. The products of 47 genes were identified, with only 11 examples predicted to be secreted proteins. Mutation in bldA, previously known to impair the stationary phase processes of antibiotic production and morphological differentiation, also induced changes in the extracellular proteome, revealing even greater pleiotropy in the bldA phenotype than previously known. Four proteins increased in abundance in the bldA mutant, while the products of 11 genes, including four secreted proteins, were severely down-regulated. Although bldA encodes the only tRNA capable of efficiently translating the rare UUA (leucine) codon, none of the latter group of genes contains an in-frame TTA. SCO0762, a serine-protease inhibitor belonging to the Streptomyces subtilisin inhibitor family implicated in differentiation in other streptomycetes, was completely absent from the bldA mutant. This dependence was shown to be mediated via the TTA-containing regulatory gene adpA, also known as bldH, a developmental gene that is responsible for the effects of bldA on differentiation. Mutation of the SCO0762 gene abolished detectable trypsin-protease inhibitory activity but did not result in any obvious morphological defects.     

A large-scale analysis of codon usage bias in 4868 bacterial genomes shows association of codon adaptation index with GC content,protein functional domains and bacterial phenotypes

Multiple synonymous codons code for the same amino acid, resulting in the degeneracy of the genetic code and in the preferred used of some codons called codon bias usage (CBU). We performed a large-scale analysis of codon usage bias analysing the distribution of the codon adaptation index (CAI) and the codon relative adaptiveness index (RA) in 4868 bacterial genomes. We found that CAI values differ significantly between protein functional domains and part of the protein outside domains and show how CAI, GC content and preferred usage of polymerase III alpha subunits are related. Additionally, we give evidence of the association between CAI and bacterial phenotypes.     

Lectin genes in the <Emphasis Type="Italic">Frankia alni</Emphasis> genome

Frankia alni strain ACN14a’s genome was scanned for the presence of determinants involved in interactions with its host plant, Alnus spp. One such determinant type is lectin, proteins that bind specifically to sugar motifs. The genome of F. alni was found to contain 7 such lectin-coding genes, five of which were of the ricinB-type. The proteins coded by these genes contain either only the lectin domain, or also a heat shock protein or a serine-threonine kinase domain upstream. These lectins were found to have several homologs in Streptomyces spp., and a few in other bacterial genomes among which none in Frankia EAN1pec and CcI3 and two in strain EUN1f. One of these F. alni genes, FRAAL0616, was cloned in E. coli, fused with a reporter gene yielding a fusion protein that was found to bind to both root hairs and to bacterial hyphae. This protein was also found to modify the dynamics of nodule formation in A. glutinosa, resulting in a higher number of nodules per root. Its role could thus be to permit binding of microbial cells to root hairs and help symbiosis to occur under conditions of low Frankia cell counts such as in pioneer situations.     

Correlation Between Shine–Dalgarno Sequence Conservation and Codon Usage of Bacterial Genes

In this study, we analyzed the correlation between codon usage bias and Shine–Dalgarno (SD) sequence conservation, using complete genome sequences of nine prokaryotes. For codon usage bias, we adopted the codon adaptation index (CAI), which is based on the codon usage preference of genes encoding ribosomal proteins, elongation factors, heat shock proteins, outer membrane proteins, and RNA polymerase subunit proteins. To compute SD sequence conservation, we used SD motif sequences predicted by Tompa and systematically aligned them with 5′UTR sequences. We found that there exists a clear correlation between the CAI values and SD sequence conservation in the genomes of Escherichia coli, Bacillus subtilis, Haemophilus influenzae, Archaeoglobus fulgidus, Methanobacterium thermoautotrophicum, and Methanococcus jannaschii, and no relationship is found in M. genitalium, M. pneumoniae, and Synechocystis. That is, genes with higher CAI values tend to have more conserved SD sequences than do genes with lower CAI values in these organisms. Some organisms, such as M. thermoautotrophicum, do not clearly show the correlation. The biological significance of these results is discussed in the context of the translation initiation process and translation efficiency. Received: 22 June 2000 / Accepted: 18 October 2000     

A Deviant Genetic Code in the Reduced Mitochondrial Genome of the Picoplanktonic Green Alga Pycnococcus provasolii

Reduction in size of flagellated chlorophytes occurred multiple times during evolution, providing the opportunity to study the consequences of cell reduction on genome architecture. Recent investigations on the chloroplast genomes of the tiny prasinophyceans Ostreococcus tauri (Mamiellales), Micromonas sp. RCC299 (Mamiellales), and Pycnococcus provasolii (Pseudocourfieldiales) highlighted their extreme compaction and reduced gene repertoires. Genome compaction is also exemplified by the Ostreococcus and Micromonas mitochondrial DNAs (mtDNAs) although they have retained almost all of the about 65 genes presumably present in the mitochondria of ancestral prasinophyceans. In this study, the mitochondrial genome of Pycnococcus was sequenced and compared to those of previously examined chlorophytes. Our results document the first case where cellular reduction of a free-living alga was accompanied by marked reduction in gene content of both the mitochondrial and chloroplast genomes. At 24,321 bp, the intronless Pycnococcus mitochondrial genome falls within the lower size range displayed by green algal mtDNAs. The 36 conserved genes, specifying two rRNAs with conventional structures, 16 tRNAs and 18 proteins, are all encoded on the same DNA strand and represent 88% of the genome. Besides a pronounced codon bias, the protein-coding genes feature a variant genetic code characterized by the use of TGA (normally a stop codon) to code for tryptophan, and the unprecedented use of TTA and TTG (normally leucine codons) as stop codons. We conclude that substantial reduction of the mitochondrial genome occurred in at least three independent chlorophyte lineages and that this process entailed a number of convergent changes in these lineages.